The present invention is concerned with a novel process for the purification and crystallization of riboflavin for pharmaceutical and foodstuff applications.
The riboflavin commercially available today is produced partly synthetically and partly biotechnologically, with the biotechnological production processes being in ascendance in recent years. Especially in the fermentative production process the purification and concentration of the riboflavin to an extent as complete as possible, as required for pharmaceutical and foodstuff applications, is extremely difficult. For such applications riboflavin is usually dissolved in an acidic or alkaline medium. Cell residues, proteins, peptides and amino acids, which, depending on the substance, can be present in dissolved or undissolved form after the dissolution of the riboflavin itself, can frequently be separated only with a relatively large effort by the combination of several different specific operations. The dissolved riboflavin is usually crystaflized out by different procedures mainly at above 30°C as needle-shaped crystals which normally correspond to the stable modification A (see, for example, US Patents 2,324,800, 2,797,215 and 4,687,847). Furthermore, riboflavin has hitherto been produced and marketed exclusively in the stable crystal modification A. As riboflavin in this form is soluble in water only to a very limited extent, the solubility behaviour necessary for pharmaceutical and foodstuff applications is relatively poor. Accordingly, for a long time there has existed the need to improve the solubility behaviour and at the same time also the bioavailability of riboflavin.
Various reports in the literature are concerned with different stable crystal modifications of riboflavin, which are formed by precipitation from alkaline solution; from such reports, however, no practical operating processes have hitherto been developed, which is presumably due to the chemical degradation of the riboflavin in alkaline medium (see, for example, US Patent 2,603,633).
The riboflavin marketed today is present partly in the form of very fine powder and partly in the form of long yellow needles. The fine powder has a considerable dustiness, an extremely low bulk density and a poor flow behaviour and becomes charged very readily, so that, for example, the pressing to tablets is hindered and additives are required to improve the flow and compacting behaviour. Likewise, the needles show a strong dust Pa/28.7.99

generation when processed and are problematic during further processing, such as, for example, in the vitaminization of flour. Also, various agglomeration procedures carried out during the crystallization have hitherto not been used for the large scale production of riboflavin (see, for example, Canadian Patent 633,852 and European Patent 307,767). Additional agglomeration procedures are carried out during drying using needle-shaped crystals of modification A (German Offenlegungsschrift 4,014,262). Accordingly, the need continues to exist to produce a form of riboflavin which possesses substantially better physical properties, such as better flow and dissolution properties and abrasive resistance, and which has a purity (riboflavin content) of above 98%.
The object of the present invention is to manufacture a purer riboflavin starting from needle-shaped riboflavin, which latter riboflavin corresponds to the stable modification A and which has been produced synthetically or biotechnologically, with a riboflavin content above 98% and which clearly possesses better flow and dissolution properties than the material currently available. In general, the new riboflavin should have an improved bioavailability and improved physical properties, e.g. in tabletting.
A relatively simple process which permits the production of such a riboflavin has now been found. This process comprises basically a so-called pre-purification and a crystalhzation; the crystallization is followed by a drying.
The process in accordance with the invention is a process for the purification and crystalhzation of riboflavin, which process comprises dissolving needle-shaped riboflavin of the stable modification A in an aqueous mineral acid solution at a temperature not exceeding about 30°C with intensive intermixing, adding active charcoal to the resulting solution, after adsorption of the dissolved impurities from the solution on the active charcoal subjecting the medium containing the active charcoal to a counter-current filtration over a ceramic membrane having a pore size of about 20 to about 200 nm, treating the resulting filtrate with a five- to ten-fold amount (vol./vol.) of water at a temperature not exceeding about 30oC and separating the resulting precipitated, spherical crystals of riboflavin by centrifugation or filtration.
After the riboflavin crystals have been obtained in this manner, the crystals can, if desired, be washed with water and subsequently dried according to methods known per se. The thus-extended process represents a further aspect of the process in accordance with the invention defined above.

The starting material used for the process in accordance with the invention is needle-shaped riboflavin of modification A, as is obtained, for example, in the production for foodstuffs. This riboflavin generally has a content of about 85 up to about 98% and, depending on its method of manufacture, chemical byproducts and/or fermentation residues as well as water, the total amount of which is accordingly above 2 weight percent.
In the first stage of the process the starting material in dry or filter-moist form is dissolved in the aqueous mineral acid solution. Thereupon, the dissolution takes place by a protonation reaction. In the dissolution procedure fermentation residues, such as proteins, peptides and amino acids, and/or chemical byproducts become liberated and are then present partly dissolved and partly in solid form. As the mineral acid there is espe¬cially suitable hydrochloric acid or nitric acid, preferably the former, the concentration of which is generally about 10% to about 65% {weight percent).
In the case of the preferred aqueous hydrochloric acid solution the concentration conveniently lies in the range of about 18 to about 24%. Up to about 19% dry riboflavin is dissolved in such an aqueous hydrochloric acid solution. The solution is thus almost saturated. In general, the amount of riboflavin relative to the amount of aqueous mineral acid depends on the nature of the mineral acid, the concentration of the solution and the dissolution temperature.
Moreover, the dissolution of the needle-shaped riboflavin in the aqueous mineral acid solution is carried out at temperatures up to a maximum 30oC, usually at about 5 to about 25oC, preferably at about 10 to about 20oC, conveniently with intensive intermixing, for example by intensive stirring.
The dissolution time can be reduced by increasing the temperature and/or the intermixing. The overall dissolution procedure generally takes up to about 30 minutes depending on the temperature and degree of intermixing.
In the next stage of the process, active charcoal is added to the solution of the riboflavin in the aqueous mineral acid solution. Consequently, the impurities present in dissolved form are adsorbed on the active charcoal. This can be powdered or granulated. Conveniently, about 0.5 to about 9% (weight percent), preferably about 3%, of active charcoal based on the riboflavin content is added for the adsorptive removal of the dissolved impurities. Depending on the impurities, the active charcoal is left in the solution for up to about 12 hours, preferably about 0.5 to about 3 hours. Acid-washed

active charcoal with a bulk density of about 250 to about 400 kg/m, preferably about 300 kg/m3 a specific surface area of about 1200 to about 1600 mVg, preferably about 1400 m /g, and an average particle size of about 20 to about 70 µm is suitable as the active charcoal. Examples of active charcoals are Norit® CAl and Bentonorit ® CAl, which are especially suitable for the adsorption of dissolved biological impurities, as well as Norit® SX2, which in turn is especially suitable for the separation of chemical impurities.
If desired, in addition to active charcoal there can be added to the aqueous mineral acid solution a filter aid, of which conveniently about 2 to about 9 weight percent based on the riboflavin content are used. Suitable filter aids are, for example, Arbocel® BWW 40 and B 800 from Rettenmaier & Sohne GmbH + Co.
The separation of the active charcoal, of the filter aid which may be present and of the undissolved fermentation residues present is carried out by the subsequent counter-current filtration. It has surprisingly been found that, in addition to the adsorption, the active charcoal also exhibits an abrasive action on the covering layer which forms on the membrane. By this action it is now possible to operate the membrane in a stable manner over a longer period of time with almost double the throughput than without active charcoal. The active charcoal thus possesses not only abrasive, but also adsorptive properties. The counter-current filtration is carried out over a ceramic membrane which has a pore size of about 20 to about 200 nm, preferably about 50 nm. The active charcoal pumped around in the circuit brings about by the abrasion a cleansing of the covering layer of carbon and fermentation residues formed on the membrane. As a rule, the counter-current velocity over the membrane is relatively high; it conveniently lies in the region of about 5 to about 6 m/s. In order not to compress the covering layer excessively, the trans-membrane pressure is conveniently 1 to 2 bar (0.1 to 0.2 MPa).
After the counter-current filtration the solution of riboflavin, which is almost free of all impurities, the active charcoal as well as filter aid which maybe present, is caused to crystalhze, which is effected by the addition of a five- to ten-fold amount of water. The resulting deprotonization of the riboflavin present in the aqueous solution leads to its precipitation.
The temperature of the medium in which the crystallization takes place can be varied in a range of 0 to 30''C depending on the production method and degree of impurity of the riboflavin. Especially in the case of synthetically produced material the temperature can be increased to 30°C; in the case of fermentative or relatively clean material

temperatures below 10°C are generally sufficient. Preferably, however, a temperature between 4 and 10°C is selected. The crystallization can be carried out batchwise or continuously, preferably continuously. Cascades or individual vessels can be used as the crystallizer. Especially in the case of individual vessels it is advisable to introduce the solution of riboflavin at different positions in the vessel. Within the crystallizer a very good macroscopic intermixing must be set up in every case. This can be realized, for example, by using a two-stage stirring device, with the feed solutions displaced by 180° being fed on to the upper and lower stirrer levels. Conveniently, for achieving this, water is added at the upper level and the mineral acid solution of the riboflavin is added at the lower level. The stirring should be carried out very carefully in order not to damage the crystals. The residence lime is suitably between about 5 and about 20 minutes, preferably about 10 to 13 minutes. The subsequent filtration is carried out using a filter or a centrifuge; there is preferably used a band filter on which also the washing, which may be carried out, is very efficient. The drying can be carried out in a manner known per se.
The initial relative supersaturation in the crystallizer (prior to the addition of water) can be regulated by recycling the mother liquor from the washing with the water flowing into the crystallizer. The ratio mother liquor:water is conveniently about 1:1 to about ] :8. The relative supersaturation can be estimated via the conductivity present in the crystallizer, with a range of about 170 to about 222 mS/cm ideally being adhered to. Depending on the conductivity the recycling of the mother liquor can be dispensed with. In the case of the recycling, it is preferably regulated through the conductivity existing in the crystallizer.
It has now surprisingly been found that by a suitable choice of mixing ratio, temperatures, intermixing and residence time it is possible in the crystallization stage of the process in accordance with the invention to crystallize a more unstable modification of riboflavin, which is spherical with a spiky surface and thus has a substantially larger surface area than the known needle-shaped crystals of modification A. Surprisingly, the spherical crystal does not result by an agglomeration procedure as has hitherto been generally described in the literature for spherical crystals [seer, for example, European Patent 307,767 and Can. J. Chem. Eng. 47, 166-170 (1969)]; on the contrary, growth of needle-shaped crystals in the case of the new process is from an initially crystallized-out, small, probably amorphous, crystal seed. The thus-obtained dendritic crystals correspond to the more soluble modification B or C, have an adequate storage stability and, furthermore, because of the more unstable modification and larger surface area, have outstanding dissolution properties and, by virtue of their spherical shape, outstanding flow properties.

Moreover, the process in accordance with the invention affords riboflavin crystals with a, higher abrasion resistance than in the case of agglomerates.
As mentioned above, the crystallizate is separated by filtration or centrifugation. The filter cake is then preferably washed with water, whereafter the moist filter cake can be dried.
The process in accordance with the invention is illustrated by the following Examples,
Example 1
The starting material used for the process described hereinafter was fermentatively produced riboflavin which had a riboflavin content of 97.02% (according to HPLC), a residual moisture content (H2O) of0.80%as well as an amino acid content of 1.11% and which was present as needle-shaped crystals of the stable modification A.
350.0 g of this starting material were dissolved in 1708.6 g of 24% hydrochloric acid at 22°C while stirring. After a dissolution period of about 15-20 minutes a brown-black solution containing about 17% of riboflavin was present.
16 g (about 3% of the amount of riboflavin) of active charcoal (Norit® CAl) were subsequently added to the solution and the mixture was stirred for a further 4 hours. The mixture was filled into the double-jacketed feed tank of a laboratory membrane apparatus. The tank was cooled in order to maintain a maximum temperature of 35°C. Using a centrifugal pump the solution was pumped over a ceramic membrane with an effective surface area of 0.0055 m2. The trans-membrane pressure was adjusted to 1.5 bar (0.15 MPa), the counter-current velocity over the membrane was adjusted to 6 m/s. This gave a permeate throughput of about 100 1/m /h, which could be maintained almost to the end of the filtration.
The solution of riboflavin in hydrochloric acid was then crystallized in a continuously operating precipitation crystallizer.
The 3 1 precipitation crystallizer was firstly filled with about 21 of water and the liquid was stirred at 100 rpm with a two-stage inclined flat blade paddle stirrer and subse¬quently cooled to 10°C. Thereafter, at about 10°C 1590 g/h of the solution of riboflavin in

hydrochloric acidic were continuously dosed in at the upper stirrer and simultaneously and continuously about 9000 g/h of water were dosed in at the lower stirrer. About 2-4 minutes after the start the riboflavin began to crystallize out as orange-yellow crystals. Initially the separated crystals appeared to be flocculent, but after 20-30 minutes they changed into granular particles. The crystal suspension was then drained off continuously until in the crystallizer the 3 1 mark {double jacket end) had been reached (i.e. after about 7 minutes). The valve was adjusted so that the level remained more or less at the 3 1 mark. The discharged suspension was added directly to a P3 suction filter where the solid was separated from the solution.
About 2500 ml of suspension were collected every 15 minutes and a filter cake of about 1 cm thickness was obtained. This was then washed in portions with 1300 ml of water until a pH of about 5 had been reached.
The moist, yellow crystallizate (65-75% residual moisture) was subsequently dried.
Example 2
A riboflavin solution was produced and treated with active charcoal as described in Example 1. In contrast to Example 1 the solution was purified over a membrane having a pore size of about 50 nm. The trans-membrane pressure was 1.5 to 1.7 bar (0.15 to 0.17 MPa) and the counter-current velocity was 5 to 6 m/s. This gave a permeate throughput of about 701/m2/h. The crystallization, filtration and washing were carried out analogously to Example 1. The crystallization temperature lay between 9 and 10°C and the drying was carried out in a laboratory drying oven at 100°C.
Example 3
The starting material used was chemically produced riboflavin having a content of 98%. The starting material was dissolved as described in Example 1. The counter-current filtration was carried out as described in Example 2. The crystallization was carried out at 20''C by dosing in 1030 g/h of solution of riboflavin in hydrochloric acid and 15060 g/h of water. Filtration and washing were carried out analogously to Example 1. The drying was carried out analogously to Example 2.
The results of the above three Examples are compiled in the following Table:

Table: Purities and properties of the respective dried final product

The respective missing percentage number comprises the water content and further slight impurities present.

We Claim:
1. A process for the purification and crystallization of riboflavin, which process comprises dissolving needle-shaped riboflavin of the stable modification A in an aqueous mineral acid solution at a temperature not exceeding about 30oC with intensive intermixing, adding active charcoal to the resulting solution, after adsorption of the dissolved impurities from the solution on the active charcoal subjecting the medium containing the active charcoal to a counter-current filtration over a ceramic membrane having a pore size of about 20 to about 200 nm, treating the resulting filtrate with a five- to ten-fold amount (vol./vol.) of water at a temperature not exceeding about 30oC and separating the resulting precipitated, spherical crystals of riboflavin by centrifugation or filtration.
2. A process according to claim 1, wherein the thus-obtained spherical crystals of riboflavin are washed with water and subsequently dried.
3. A process according to claim 1 or 2, wherein the mineral acid is hydrochloric acid or nitric acid, preferably hydrochloric acid.
4. A process according to any one of claims 1 to 3, wherein the dissolution of the needle-shaped riboflavin in the aqueous mineral acid solution is carried out at about 5 to about 25oC, preferably at about 10 to about 20oC.
5. A process according to any one of claims 1 to 4, wherein about 0.5 to about 9% (weight percent relative to the riboflavin content), preferably about 3%, of active carbon is added for the dissolution of the needle-shaped riboflavin in the aqueous mineral acid solution.
6. A process according to any one of claims 1 to 5, wherein acid-washed active charcoal with a bulk density of about 250 to about 400 kg/m3 preferably about 300 kg/m3, a specific surface area of about 1200 to about 1600 m2/g, preferably about 1400 m2/g, and an average particle size of about 20 to about 70 µm is used as the active charcoal.
7. A process according to any one of claims 1 to 6, wherein a filter aid is added in addition to the active charcoal.

8. A process according to any one of claims 1 to 7, wherein the ceramic
membrane has a pore size of about 50 nm,
9. A process according to any one of claims 1 to 8, wherein the temperature of
the medium in which the crystallization takes place hes in a range of about 4 to about 10oC.
10. A process according to any one of claims 1 to 9, which is carried out
continuously and wherein the residence time in the crystallizer during the crystallization is
about 5 to about 25 minutes, preferably about 10 to 13 minutes.
11. A process according to any one of claims 1 to 10, wherein the resulting
precipitated, spherical crystals of riboflavin are collected on a band filter, separated and
dried.
12. A process for the purification and crystallization
of riboflavin. substantially as herein described and
exemplified.